Jeffrey L. Platt

Prolongation of cardiac xenograft survival by depletion of complement

Complement (C) activation is thought to be critical for the hyperacute rejection of xenografts. We investigated the role of C in the rejection of discordant cardiac xenografts by studying outcome in recipients depleted of C, using a highly purified form of cobra venom factor (CVF) in both a small (guinea pig [GP]-to-rat) and large (pig-to-baboon) animal model. A single dose of 30 or 60 units CVF given i.v. to rats completely abrogated hemolytic C activity for up to 72 hr. The lack of hemolytic C activity correlated with nearly undetectable serum levels of C3. Doses of 30 U/kg daily or 60 U/kg every other day over a 7-day period sustained C depletion without morbidity or mortality. Rats receiving GP cardiac xenografts during CVF therapy had significantly prolonged xenograft survival (88 +/- 10 hr in CVF-treated rats vs. 18.6 +/- 7.2 min in control rats, P < 0.001). Rats that rejected GP xenografts at 4 days posttransplant had higher levels of anti-GP antibodies than control rats, without hemolytic C activity at rejection. This rise in xenoreactive Ig reflected an increase in circulating IgG and IgM against GP antigens recognized before transplantation. Histologic analysis of GP cardiac xenografts taken from CVF-treated rats revealed leukocyte and monocyte margination along blood vessels, beginning at 12 hr posttransplant. Progressive cell infiltration, interstitial hemorrhage, and necrosis were observed over the next 72 hr. Rejected GP xenografts showed diffuse deposition of IgM and fibrin within blood vessels but no evidence of C3 deposition. A nonspecific pattern of IgG deposition was noted. CVF was tested in baboons. Complete C depletion was achieved with a dose of 60 U/kg, and was not associated with any morbidity or mortality. Xenotransplantation of a pig heart was performed in one baboon receiving CVF, 60 U/kg/day, for 2 consecutive days. Xenograft survival was prolonged to 68 hr, compared with 90 +/- 30 min in control baboons. Lack of hemolytic activity was noted during engraftment and at rejection. Histology showed evidence of vascular rejection. Immunopathology showed diffuse deposition of IgM, fibrin, and C4, and absence of C3 or membrane attack complex. We conclude that highly purified CVF can achieve marked C depletion with minimal morbidity and no associated fatalities. CVF alone can significantly prolong discordant cardiac xenograft survival. In the GP-to-rat model, the improvement in graft survival achieved with CVF was better than with conventional immunosuppression or isolated acute antibody depletion.(ABSTRACT TRUNCATED AT 400 WORDS)

Inhibition of complement-mediated endothelial cell cytotoxicity by decay-accelerating factor: Potential for prevention of xenograft hyperacute rejection

Complement plays a major role in hyperacute rejection of discordant xenografts. In immediately vascularized xenografts, such as porcine organs to humans, C activation contributes to triggering of endothelial cell activation and adhesion of leukocytes and platelets to the endothelial cells, which is followed by thrombosis and tissue necrosis. We investigated the potential utility of the membrane-associated inhibitor of C, decay accelerating factor (DAF), in the prevention of C-mediated tissue injury. We used an in vitro model of xenotransplantation consisting of porcine aortic endothelial cells incubated with human serum as the source of xenogeneic natural antibodies and C. Because C inhibitors such as DAF may be relatively species-specific, we tested whether human DAF would incorporate into porcine endothelial cells and function to inhibit cytotoxicity of such cells by human C. We found that purified radiolabeled human DAF incorporated into porcine endothelial cells in a dose-dependent manner and that human DAF very significantly protected the endothelial cells from the cytotoxic effect of human C.

Vascular endothelial cell proteoglycans are susceptible to cleavage by neutrophils

Heparan sulfate proteoglycan associated with vascular endothelial cells in vivo plays an important role in a number of endothelial functions, including the inhibition of intravascular coagulation and extravasation of plasma proteins and blood cells. In this report, we demonstrate that polymorphonuclear neutrophils, as well as cell-free neutrophil supernatants, lead to a rapid and dramatic loss of proteoglycan from endothelial cells in the absence of evidence of cell lysis. This cleavage appears to be relatively (although not absolutely) selective for heparan sulfate and is mediated by neutrophil-derived serine proteases. Inhibitors of neutrophil elastase appear to be the most effective inhibitors of proteoglycan release. Furthermore, purified human neutrophil elastase also leads to cleavage of cellular proteoglycans, although not to the same extent as neutrophils or neutrophil supernatants. Proteoglycans compared with all other protein-containing macromolecules appear to be especially vulnerable to neutrophil-mediated cleavage. The results of this study may be germane to the interaction of neutrophils with endothelium during the inflammatory process, during which the loss of endothelial heparan sulfate proteoglycan may play a critical role in the vascular injury that often accompanies inflammation.

Effect of T cell modulation on the translocation of bacteria from the gut and mesenteric lymph node

Although the ability of the gut-associated lymphoid tissue (GALT) to respond to orally ingested foreign antigens has been studied extensively, its function in preventing or limiting escape of resident gut bacteria has not been assessed. The following studies were performed to examine what role cell-mediated immunity (CMI) plays in this process. The ability of suppression of CMI to induce escape of gut bacteria (translocation) to the mescnteric lymph node (MLN) in immunocompctent mice whose gut flora was unaltered was examined. Administration of cyclosporine or anti-L3T4 antibody failed to induce translocation of indigenous gut bacteria after 7 or 14 days of treatment. Antithymocyte globulin (ATG) also failed to induce translocation after 7 days of treatment, despite depletion of all Thy 1, Lyt 1, L3T4, and Lyt 2 positive cells from the spleen, MLN, and intestine as demonstrated by immunofluorescent microscopy. Finally, cultures of the MLN, spleen, liver, and peritoneum of T cell-deficient BALB/c nude mice and their heterozygous T cell-replete littermatcs were also sterile, demonstrating that congenital suppression of T CMI also docs not lead to translocation of indigenous gut bacteria. The role of CMI in limiting systemic spread of bacteria that were already translocating to the MLN was also examined. Translocation of Escherichia coli C25 to the MLN was induced by gastrointestinal (GI) monoassociation, which leads to translocation of E. coli C25 to the MLN in 80–100% of mice. Treatment with ATG during monoassociation failed to induce spread of E. coli C25 to the spleen, liver, or peritoneum, despite the same degree of T cell depletion achieved with ATG in the previous experiment. Monoassociation of conventional T cell-deficient BALB/c nude and hetero/ygous mice and germfree T cell-deficient BALB/c nude and heterozygous mice also did not lead to spread of E. coli C25 beyond the MLN. However, in ATG-treated, conventional nude, and germ-free nude mice, the average number of translocating E. coli C25 per MLN was consistently higher. In separate experiments the ability of stimulation of T cell function to inhibit translocation of E. coli C25 was examined. Rccombinant intcrleukin-2, 25,000 units, was administered intraperitoneally every 8 hours during exposure to E. coli C25. This reduced the incidence of translocation of E. coli C25 from 85% to 51% (p = 0.02). Suppression of CMI, either systemically or within the GALT, has n minimal influence on the mechanisms by which the normal gut flora are translocated to the MLN. However, suppression of CMI promotes increased survival of the bacteria that have translocated to the MLN. Conversely, augmentation of T cell function may reduce the number of bacteria that survive in the node, possibly by enhanced bacterial killing within the local environment of the MLN.

Evidence that 15-deoxyspergualin inhibits natural antibody production but fails to prevent hyperacute rejection in a discordant xenograft model

Preventing hyperacute rejection (HAR) is a difficult and unsolved problem in xenotransplantation. This may be due, in part, to a lack of therapies that can suppress production of natural antibodies (NA), which are thought to be critical mediators of HAR. This study examined the effect of 15-deoxyspergualin (DSPG) and splenectomy (Spx) on NA production and return of NA after plasma exchange (PE) in a discordant species combination (strain 2 guinea pig to Lewis rat). A dose of 5 mg/kg/day DSPG + Spx significantly reduced Lewis rat anti-guinea pig NA titer after one week of therapy. Antibody titer was not significantly reduced in rats treated with splenectomy alone. PE alone acutely depleted NA titers; however, complete rebound was seen in 48 hr. When PE was performed in rats treated with DSPG + Spx, an additional significant NA reduction occurred; no rebound 24-48 hr after PE was seen. Except for a 20% reduction in body weight, no serious complications occurred in DSPG + Spx recipients. Despite a profound NA titer reduction, treatment with DSPG, Spx, and PE did not prolong guinea pig cardiac xenograft survival in a clinically significant fashion. Immunopathological study of rejected cardiac xenografts revealed no antibody deposition but persistent complement deposition on vascular endothelium. We conclude that DSPG + Spx effectively inhibits synthesis of rat anti-guinea pig NA, that further NA titer reduction can be achieved with the addition of PE, and that DSPG + Spx prevents post-PE antibody rebound. We also conclude that the limited prolongation in cardiac xenograft survival achieved, despite marked suppression of NA, supports a complement-mediated mechanism of HAR in our animal model.

Cell populations and membrane attack complex in glomeruli of patients with post-streptococcal glomerulonephritis: Identification using monoclonal antibodies by indirect immunofluorescence

Poststreptococcal glomerulonephritis (PSGN) had been thought to arise from renal deposition of immune complexes and as such is analogous to acute serum sickness. Recent studies of acute serum sickness in animals and PSGN in humans, however, have suggested a pathogenetic role for cellular immunity. To enlarge on these observations, cellular components of glomeruli were characterized by indirect immunofluorescence in 11 tissues from individuals with PSGN using monoclonal antibodies. These studies demonstrate infiltration of glomeruli by monocytes, granulocytes, and lymphoid cells. Focal accumulations of T lymphocytes were also observed adjacent to Bowmans capsule. Analysis of glomerular T-cell subpopulations revealed a predominance of cells reactive with OKT4 early and with OKT8 later in the course of disease. Proliferation of parietal and visceral epithelial cells was associated with increased binding of BA-1 and J5, respectively. The presence of the membrane attack complex of complement was demonstrated by glomerular reactivity with a monoclonal antibody (poly-C9 MA) which recognizes a neoantigen present in poly-C9. Fluorescence was present along the glomerular basement membrane early and within the mesangium late in the course of disease, a distribution similar to that observed for C3 and C5. These observations suggest that immune cells as well as terminal components of complement either provoke or mark tissue injury in PSGN.

Heparin and heparan sulfate delimit nephron formation in fetal metanephric kidneys

Formation of nephrons from primitive mesenchyme in fetal kidneys is induced by ureteric buds. Nephron induction is closely coordinated with branching morphogenesis of the ureteric bud. Having previously shown that branching of the primitive ureter is associated with de novo synthesis of chondroitin sulfate proteoglycan and release of free heparan sulfate glycosaminoglycan chains, we asked whether glycosaminoglycans influence nephron development. Fetal mouse kidneys were incubated in organ cultures containing heparan sulfate, heparin, chondroitin sulfate, or hyaluronate. After 48 hr the number of nephrons at each developmental stage was enumerated by light microscopic analysis of serial tissue sections. Kidneys incubated in heparin or in heparan sulfate contained up to 10-fold fewer nephrons than did kidneys incubated in control conditions or in chondroitin sulfate or hyaluronic acid. Maturation of nephrons, however, was unaffected. Inhibition of nephron development was associated with binding of labeled heparin to primitive mesenchyme and altered tissue distribution of fibronectin. Branching morphogenesis was impaired in kidneys exposed to heparin but not to heparan sulfate or to de-N-sulfated, N-acetylated heparin. The capacity of glycosaminoglycans to inhibit nephron formation depended on sugar composition and O-sulfation but not GAG chain size or charge density. Thus, heparan sulfate may have the capacity to specifically control formation of nephrons in fetal metanephric kidneys in vitro.

Suppression of synthesis of natural antibodies by mycophenolate mofetil (RS-61443). Its potential use in discordant xenografting.

One of the major barriers to successful transplantation of immediately vascularized organs between discordant species is the presence of natural antibodies (NA) in the recipient. While natural antibodies can be depleted by plasmapheresis and/or organ absorption, they rapidly return to the circulation after such procedures. It will be desirable to suppress NA for longer periods of time. Since NA appear to be produced at least in part by CD5+ B cells, it was important to evaluate whether mycophenolate mofetil (RS-61443), a novel immunosuppressant that has been shown to suppress normally elicited antibody synthesis, would also be able to suppress NA. Adult rats were splenectomized, and 2 days later, 9 plasma exchanges, each of 4 ml, were performed. One group of rats received RS-61443 at 40 mg/kg/day (the dose described as efficacious for suppressing elicited antibodies in rats) starting immediately after the last exchange for 7 days, and then 20 mg/kg/day for an additional 7 days; no drug was given to the control group. NA levels were measured at various times by ELISA, using guinea pig platelets extracts as the target. Splenectomy alone led to a significant decrease from the control levels of NA; liters were further reduced by the plasma exchanges. In the absence of RS-61443, NA titers rose steadily, starting at 24 hr after the last plasma exchange. In contrast, administration of RS-61443 resulted in levels of NA on day 7 not significantly different from those after plasma exchange, reducing the dose of RS-61443 to the 20 mg/kg/day level during week 2 allowed the gradual return of NA. Administration of RS-61443 at the 40 mg/kg/day dose to rats after splenectomy alone led to a clear and significant further decrease in NA levels over the first week. It has been shown that RS-61443 can be administered for longer periods. The data presented suggest that use of this drug, perhaps with more conventional agents, may allow suppression of NA for a significant period after transplantation.

Chondroitin sulfate proteoglycan synthesis and reutilization of β-d-xyloside-initiated chondroitin/dermatan sulfate glycosaminoglycans in fetal kidney branching morphogenesis

Branching morphogenesis and chondroitin sulfate proteoglycan synthesis by explanted fetal mouse kidneys were previously shown to be inhibited by p-nitrophenyl beta-D-xylopyranoside (beta-D-xyloside) while glomerular development and heparan sulfate proteoglycan synthesis were unaffected. The metabolic fate of fetal kidney explant proteoglycans was investigated to determine whether or not recovery of proteoglycan synthesis and morphogenesis occur after exposure to beta-D-xyloside. Chondroitin sulfate proteoglycan synthesis resumed within 4 hr of removal of beta-D-xyloside and was enhanced once beta-D-xyloside-initiated chondroitin/dermatan-35SO4 glycosaminoglycans (GAGs) were released from the tissue. Radioactivity incorporated into beta-D-xyloside-initiated chondroitin/dermatan-35SO4 GAGs during labeling in the presence of beta-D-xyloside was reutilized in the synthesis of chondroitin-35SO4 proteoglycan during a 24-hr chase in nonradioactive medium without beta-D-xyloside. Further, highly purified beta-D-xyloside-initiated chondroitin/dermatan-35SO4 GAGs were taken up by kidneys more avidly than was free [35S]sulfate. These 35S-GAGs were degraded and reutilized in the synthesis of chondroitin-35SO4 proteoglycan. Ureteric bud branching resumed 48 hr after beta-D-xyloside was removed from the incubation medium. These findings support the idea that both chondroitin sulfate proteoglycan synthesis and proteoglycan processing may be involved in branching morphogenesis.

Rejection in single versus combined pancreas and kidney transplantation in pigs

Clinically, the incidence of reversible renal allograft rejection episodes appears to be higher in recipients of simultaneous pancreas/kidney (SPK) than of kidney transplantation alone (KTA); conversely, the rate of irreversible pancreas allograft rejection appears to be higher in pancreas transplant alone (PTA) than SPK recipients. Clinical/histological correlation of graft rejection in these three groups has not been precise. Therefore, we studied the incidence and histological severity of the rejection process in a large animal (pig) model of SPK (n = 36), PTA (n = 31), and KTA (n = 36) allotransplantation. SPK and PTA recipients were made diabetic pretransplant by streptozotocin (150 mg/kg). Pancreas graft exocrine secretions were bladder-drained via a duodenocystostomy for urine amylase (UA) monitoring; endocrine function was monitored by plasma glucose (PG) levels. SPK and KTA recipients underwent native nephrectomy, and renal allograft function was monitored by serum creatinine (CR). Cyclosporine, azathioprine, and prednisone were given in tapering doses from the time of transplantation. Grafts were biopsied weekly to grade histologic severity of interstitial and vascular rejection on light microscopy (LM) and for intensity of T cell infiltration on immunofluorescence. Pancreas graft exocrine function (UA above pretransplant baseline), present in 62% of PTA and 68% of SPK recipients at one week, persisted in only 7% of PTA vs. 64% of SPK pigs at 2 weeks (P = 0.0004). Likewise, pancreas graft endocrine function (PG < 200 mg/dl off insulin) was sustained longer in SPK than PTA recipients (100% vs. 84% at 1 and 91% vs. 27% at 2 weeks; P = 0.0006). However, renal allograft functional survival (serum creatinine < 3.0 mg/dl) was not significantly different (P = 0.471) between SPK and KTA recipients (36% vs. 30% at 1 and 23% vs. 13% at 2 weeks). Graft functional parameters partially correlated with biopsy observations. Pancreas allograft biopsies showed a significantly (P = 0.03 at 1 and P = 0.05 at 2 weeks) lower incidence of moderate/severe interstitial rejection in SPK than PTA recipients (67% vs. 95% at 1 and 57% vs. 92% at 2 weeks); rejection was absent in 8% of SPK and in no PTA biopsies at 1 week. Vascular rejection was moderate/severe in significantly fewer (P = 0.0013 at 1 and P = 0.023 at 2 weeks) SPK than PTA pancreas grafts (13% vs. 37% at 1 and 14% vs. 38% at 2 weeks).(ABSTRACT TRUNCATED AT 400 WORDS)